Despite major advances in understanding the molecular and genetic basis of cancer, disease progression to metastasis remains the cause of >90% of cancer-related mortality. Understanding the mechanisms underlying metastasis initiation is critical for the development of new therapeutic strategies specifically to diagnose, target and prevent progression to metastatic disease. However, the identity of metastasis-initiating cells in human breast cancer remains elusive. We propose to use an integrative approach using a variety of state-of-the-art single cell multi-parametric, analytical techniques to probe heterogeneity during metastasis of human breast cancer at single cell resolution. We have previously found heterogeneity in micro- and macro-metastatic cells from human patient-derived xenografts (PDXs) and .we will exploit these PDXs grown in immunodeficient mice to probe heterogeneity in triple negative, estrogen receptor-positive and HER2-positive human breast cancers and their metastases. We propose to determine differentiation states for triple negative breast cancers and their early and advanced metastasis using single cell profiling techniques to test the hypothesis that distinct states confer therapeutic resistance and metastatic capabilities. We will compare the differentiation states of the triple negative cancers with those of estrogen receptor positive and HER2 positive breast cancers and their early and advanced metastases at the single cell level to determine the properties of states that confer therapeutic resistance and metastatic capabilities. The RNA studies will be complemented by protein analysis using single cell mass cytometry. After determining the self-renewal and differentiation properties of the metastatic cells, we will test their response to targeted therapy by transplantation in vivo and by tumor cell sphere assays in culture. We will then validate the predictions by examining human breast cancer specimens. Through this systems biology approach we plan to develop a global understanding of the events leading to breast cancer metastasis. Understanding these functions in molecular detail could lead to prevention regimes for patients at high risk of developing breast cancer so that their tumors do not metastasize, biomarkers of progression for patients who are newly diagnosed with cancer and new therapeutic approaches for patients at high risk of developing metastases by either suppressing the outgrowth of micro-metastases or by killing them outright.